1. Field of the Invention
This invention relates to flow cytometry and, in particular, to a method and apparatus for staining cells for subsequent analysis and/or sorting in a flow cytometer.
2. Description of the Prior Art
Flow cytometers are widely used to sort and quantify specific cell types within a population of cells. One common application of the procedure involves the quantification of lymphocyte subsets within a leukocyte population. Such quantification is of importance in various diseases, including AIDS where one of the primary tests for evaluating patients involves measuring the level of CD4+ lymphocytes in peripheral blood samples.
A flow cytometric analysis involves two basic steps: 1) staining selected cell types, and 2) determining the number of stained cells relative to the total number of cells in the population. The present invention is concerned with the staining step. Commercially available equipment, e.g., equipment manufactured by such companies as Coulter Electronics, Hialeah, Fla., and Becton-Dickinson, Mountain View, Calif., is widely available for performing the cell sorting and counting step of the analysis.
As presently practiced, staining of cells for flow cytometry is performed using antibodies, e.g., monoclonal antibodies, which recognize specific cell types within the population of cells. The antibodies are either directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescent-labeled second antibody which recognizes the first antibody. Typically, a panel of antibodies is used to analyze the cell population, with samples of the population being stained with each of the antibodies in the panel and then analyzed in the flow cytometer.
The staining of a cell population with panel of antibodies involves multiple manipulative steps. First, a series of samples from the population are prepared. Then, one antibody from the panel is combined with each of the samples, and the resulting mixture is incubated so that the antibody can bind to the cells in the sample (if any) which the antibody recognizes. One or more washings of the cells are then performed to remove unbound antibody. If the first antibody has not been directly labeled with a fluorescent compound, additional incubations with other antibodies or reagents and additional washings are in general required to label the unconjugated primary antibody bound to the cells. Finally, the stained cells are transferred to the flow cytometer for sorting and analysis.
Plainly, a procedure having these many steps is difficult to perform efficiently and accurately. Moreover, because of the high level of concentration and attention to detail that is needed to avoid mistakes, the procedure as presently practiced does not lend itself to performance by relatively unskilled technicians, as is desired when large numbers of analyses are to be performed, e.g., in a clinical setting. An error in performing an analysis, if recognized, leads to a repeated procedure with a concomitant loss of time and reagents. An unrecognized error results in invalid data which can have devastating consequences, especially where patient management is involved.
At present, there is no uniform system for staining cells for flow cytometry. Each laboratory which adopts the technique is faced with the problem o developing its own protocols for conducting the various steps in the procedure. Although extensive efforts to develop suitable protocols have been made by researchers and supervisory personnel, there still does not exist a truly efficient and easy-to-perform technique for staining cells for flow cytometry.
Examples of the techniques which have been developed can be in found in the following references:
1. Braylan, R. C., Chen, M. G., Iturraspe, J. A., and Benson, N. A., "Immunophenotyping of Leukemias and Lymphomas in Microtiter Trays," Cytometry Supplement, (1988) vol. 2, page 52. PA1 2. Edwards, B. S., and Shopp, G. M., "Efficient Use of Monoclonal Antibodies for Immunofluorescence," Cytometry, (1989) vol. 10, pages 94-97. PA1 3. Gitter, B. D., Finn, O. J., and Metzgar, R. S., "Cytofluorometric Isolation of 1937, an Ia Antigen-Bearing Variant of the Ia-Negative Human Monocytic Cell Line U937," J. of Immunology, (1985) vol. 134, pages 280-283. PA1 4. Ledbetter, J. A., Rouse, R. ".V, Micklem, H. S., and Herzenberg, L. A., T Cell Subsets Defined By Expression of Lyt-1,2,3 and Thy-1 Antigens," J. Exp. Med., (1982) vol. 152, pages 280-295. PA1 5. Lindqvist, C., Patarroyo, M., Beatty, P. G. and Wigzell, H., "A Monoclonal Antibody Inhibiting Leucocyte Adhesion Blocks Induction of IL-2 Production but not IL-2 Receptor Expression," Immunology, (1987) vol. 60, pages 579-584. PA1 6. Loken, M. R. and Stall, A. M., "Flow Cytometry as an Analytical and Preparative Tool in Immunology," J. Immunol. Methods, (1982) vol. 50, pages R85-R112. PA1 7. Muirhead, K. A., Wallace, P. K., Schmitt, T. C., Frescatore R. L., Franco, J. A., and Horan, P. K. "Methodological Considerations for Implementation of Lymphocyte Subset Analysis in a Clinical Reference Laboratory," Annals New York Academy of Sciences, (1986) vol. 468, pages 113-127. PA1 8. Rector, E., Nakajima, T., Rocha, C., Duncan, D., Lestourgeon, D., Mitchell, R. S., Fischer", J., Sehon, A. H., and Delespesse, G., Detection and Characterization of Monoclonal Antibodies Specific to IgE Receptors on Human Lymphocytes by Flow Cytometry," Immunology, (1985) vol. 55, pages 481-488.
In particular, Loken et al. describe a staining procedure in which cells are aliquoted into a microtiter plate, pelleted, and the supernatants removed by suction. Thereafter, stains are added to the wells and the cells in each row are resuspended using an 8-channel multiple micropipette. After various incubation/washing steps, the cells are transferred to test tubes using a 4-channel multipipette for analysis in a flow cytometer.
The use of microtiter plates to perform staining is also described in Muirhead et al., Lindquist et al., Ledbetter et al., Edwards et al. and Gitter et al. The Edwards and Gitter references also describe centrifuging a microtiter plate and removing the supernatant liquid from the cell pellet by a single downward jerking motion or flicking of the plate. Braylan et al. describe an automated immunofluorescence flow cytometry technique using microtiter plates. Rector et al. describe a method for detecting antibodies in hybridoma supernatants using a flow cytometer technique in which 96-well V-bottomed microtiter plates, 96-well flat-bottomed microtiter plates, and microcentrifuge tubes were used to conduct the various reactions and in which transfer between the V-bottomed and flat-bottomed microtiter plates was performed using a multichannel pipette.
The use of multichannel pipettes with microtiter plates is also shown in U.S. Pat. Nos. 3,807,235, 3,982,438, and 4,158,035. In each of these patents, the multichannel pipette is mounted on a supporting member and either the pipette is moved vertically relative to the plate or the plate is moved vertically relative to the pipette when pipetting is to performed.
In addition to the foregoing, U.S. Design Patents Nos. Des. 248,045 and Des. 250,348 show a pipette holder and a test tube rack, respectively, and U.S. Pat. No. 4,599,315 shows a microdroplet test plate and a cover for the plate, where the cover and the plate have a corresponding set of letters and numbers. Also, test tube strips and carriers for test tube strips of the type shown in FIG. 2 but without shoulders 62 and 64 are commercially available but have not been reported in the literature for use in the staining of cells for flow cytometry (see elements 74 and 82 in FIG. 2). From another commercial supplier, individual tubes have been described for holding small cell samples that are received in the glass sample chamber of an Epics C Flow Cytometer manufactured by Coulter Electronics. Such tubes, however, have not been supplied as a test tube strip in a carrier that would be conformationally compatible with a multichannel pipette and microtiter plate.